At the present time, all research in this laboratory is directly related to the persistence, pathophysiology, and detection of viruses in blood, mainly HBV and HCV, but also including some emerging infectious agents. Despite the licensure and application of sensitive serologic assays for the hepatitis B virus (HBV) and hepatitis C virus (HCV), a few cases continue to be transmitted by blood, plasma, and products made from them. Ongoing research throughout the world has led to the development of new assays and new concepts concerning these viruses, and this has made it necessary for CBER to have an active research program to evaluate the new assays. In addition, research had been necessary to try to understand why some infected individuals develop chronic infections while others recover; why some individuals develop life-threatening chronic liver disease, while others remain asymptomatic even if chronically infected; and why blood donations sometimes transmit these viruses despite the use of sensitive screening tests. In the case of HBV, the recognition of "silent" cases that cannot be detected by serologic screening tests that are currently used for whole blood donations, as well as the growing recognition that mutant forms of the virus sometimes develop that can escape immune surveillance, have provided impetus for research on this virus. This laboratory has also focused on emerging infectious agents that can be transmitted by blood, particularly those high risk agents for which the private sector has not initiated research. An example has been the studies in this lab to evaluate reports that SV40, a cancer virus in animals, was allegedly found in the blood of normal blood donors. Silent Hepatitis B Virus. This project is currently undergoing re-design to focus on silent hepatitis B virus in blood and plasma donors, i.e. those donors whose hepatitis B virus infections are not detected by current screening procedures. The project will involve a close analysis of variant (mutant) HBV strains found around the world, in order to determine the role of HBV mutations in the failure to detect occasional donors with HBV infection. Seroepidemiology and Molecular Biology of Hepatitis B and C Viruses. Research on the seroepidemiology of the hepatitis B virus (HBV) and hepatitis C virus (HCV) in serially collected serum samples from Miyazaki, Japan, has continued. One of the villages has a 23% prevalence rate of HCV infection, and studies have shown that the incidence of NEW infections in previously seronegative persons is extremely high, approaching 300 cases/100,000 person-years. Later studies in other villages elsewhere in Japan published by other laboratories have subsequently provided additional support for the concept that pockets of extremely high prevalence of HCV are probably responsible for the high rate of HCV-associated disease in Japan. HCV QUASISPECIES: The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral persistence. Changes in the components of this quasispecies population allow the virus to escape from the host immune response. To evaluate quasispecies changes, serial serum samples were studied from prospectively followed individuals in the Miyazaki Cohort Study, to observe the changes in HCV quasispecies that occurred over a period of up to 10 years. RNA was extracted from serial serum samples from six patients who subsequently developed HCV-associated liver cancer and 11 age- and sex-matched HCV-infected controls without liver cancer. The hypervariable region 1 (HVR1) of the HCV genome was amplified by RT-PCR using nested primers and then cloned. Ten clones were sequenced from each of several serial serum samples obtained over a ten-year period from each subject. In each of the sequential serum samples, a mixture of HCV quasispecies was found. The average genetic distance among 10 clones from each individual sample was determined. During the disease progression, the number of quasispecies increased. A collateral aspect of the study was an attempt to define the ideal number of clones to sequence in order to obtain maximal information about HCV quasispecies, since this has not previously been studied. The value of sequencing greater than 3 to 5 clones of HCV (the number usually reported in the literature) has never been established. To evaluate this, 12 serum samples from two liver cancer patients and two matched controls were selected, and successive groups of 10 additional clones were sequenced up to a total of 50 clones per serum sample. After having sequenced 40 clones per sample, sequencing an additional 10 clones per sample (i.e. a total of 50 clones per sample) detected only one or no additional quasispecies. Thus, we concluded that, although certain differences in HCV clones in HCV-infected patients could only be detected by testing 40 clones per serum sample, testing an additional 10 clones (50 clones per sample) did not significantly increase the number of quasispecies detected. THE SENSITIVITY OF NAT FOR HBV DNA AND ASSAYS FOR HBSAG: A small risk of transfusion transmitted HBV remains, despite the use of sensitive licensed tests for HBsAg and anti-HBc to screen blood donations. Since the year 2000, nucleic acid amplification tests (NAT) on minipools to detect HBV have been applied under INDs to almost all source plasma collected in the United States. However, minipool NAT to detect HBV in whole blood donations has not been introduced in the U.S. due to the expectation that newer HBsAg immunoassays that have been approved recently or are currently under development are of equal or greater sensitivity than minipool NAT. These newer HBsAg immunoassays also are more sensitive than most of the HBsAg tests currently licensed in the U.S. The newer HBsAg immunoassays can detect HBsAg in any infected unit containing about 400 HBV DNA IU/ml, and in some cases they can detect samples with even lower levels. This laboratory designed and participated in a collaborative study to determine the relative sensitivities of currently licensed HBsAg tests, newer more sensitive HBsAg tests that are under development, minipool NAT for HBV DNA, and single-donor NAT for HBV DNA. In this study, 100 representative serum samples were selected from 10 seroconversion panels and coded along with 28 analytical controls (FDA HBsAg lot-release panel and dilutions of the WHO HBV NAT standard). All 128 samples were tested using seven HBsAg tests (some currently licensed and some currently under development), and by several versions of NAT to detect HBV DNA (four minipool and three single-donor formats). HBsAg concentrations at cut-off for the newer HBsAg tests ranged from 0.07-0.12 ng/mL, compared with 0.13-0.62 ng/mL for currently licensed HBsAg tests. The HBV DNA concentration at the point of HBsAg cut-off ranged from 102-267 IU/mL for the newer HBsAg tests, and from 363-1069 IU/mL for currently licensed HBsAg tests. The newer HBsAg tests would be expected to detect additional infected units compared to licensed HBsAg tests, and the newer HBsAg assays and minipool NAT assays were of comparable sensitivity. However, single-donor NAT would increase the detection of HBV-containing samples even further. THE RELATIVE LEVELS OF HBV DNA AND INFECTIVITY IN HUMAN PLASMA: Infectious inocula derived from human serum samples containing HBV, for which infectivity titers had been studied in chimpanzees, were evaluated using TaqMan to determine the relationship between virus copy number and infectivity. The genotypes of the inocula containing the HBV adw, ayw, and adr subtypes were determined to be A, D, and C, respectively. Using standard curves created from several materials (linear within the range of 101 to 108 genome equivalents [geq] per ml) and testing several stored standard dilutions of the inocula, it was estimated that the original undiluted BoB/NIAID inocula of adw subtype contained 5.3x10e9 geq/ml, of the ayw subtype contained 2.5x10e9 geq/ml , and of the adr subtype contained 2.7x10e8 geq/ml. Based on earlier studies showing that the dilution end-points of infectivity for these inocula in chimpanzees were 10e-7/ml for the adw inoculum, 10e-7.5/ml for the ayw inoculum, and 10e-8/ml for the adr inoculum, the minimum infectious dose in chimpanzees for subtype adw was estimated to be 540 geq (about 90 IU), for the ayw subtype to be 80 geq (about 13 IU), and for the adr subtype to be 3 geq (0.5 IU) (although results for the adr subtype are still undergoing further investigation). ASSAY FOR HEPATITIS C CORE ANTIGEN: The detection of HCV core protein was compared with the detection of HCV RNA to identify the presence of viremia in individuals with antibody to HCV (anti-HCV). Ninety adults in Miyazaki Prefecture, Japan, who had detectable anti-HCV in their serum, were studied. Among anti-HCV-positive individuals in this population, 84% were HCV RNA-positive, and 86% of those were also positive for HCV core protein. The detection of HCV core protein may be a useful alternative to the detection of HCV RNA for the identification of active HCV infection in some situations. Emerging Infectious Diseases. SV40 VIRUS: SV40, a polyomavirus, is a small non-enveloped DNA virus that has been detected in several human brain tumors as well as in a number of animal tumors. In 2002, it was reported that SV40 sequences could be detected in the buffy coat cells from 29% of normal blood donors using PCR for five segments of the virus; about the same time, additional reports appeared in the literature stating that SV40 could be detected in non-Hodgkin lymphoma. These reports, combined with the knowledge that millions of Americans received poliovaccine in the 1950s and 1960s that was inadvertently contaminated with SV40, suggested that SV40 could pose a danger for blood recipients. However, the report of SV40 in buffy coat cells had several significant shortcomings. We undertook to try to duplicate the finding of SV40 in buffy coats from normal blood donors. We wanted to do this with a study design that would not have the weaknesses of the published study; for this purpose, we designed the study to eliminate the possibility that other ubiquitous polyomaviruses, BK and JC viruses, could have been responsible for any positive results. We also conducted the study using two separate methods, PCR and TaqMan. The objectives of this study were to look for the SV40 genomic regions corresponding to the TAg NH2 terminus, COOH terminus, the VP-1 structural protein, and the regulatory region in the buffy coats of 200 blood donors. Samples were first studied for BK and JC DNA. They were tested subsequently by both PCR and TaqMan for the four SV40 sequences. Samples positive for SV40 DNA were further investigated to exclude the possibility of laboratory contamination. All of these studies are still in progress. SEN-V VIRUS: The aim of this study was to evaluate the prevalence of two variants of SEN-V virus in serum and to use the HCC and liver tissue from these patients to try to develop an in situ hybridization assay for SEN-V. The ultimate purpose was to try to identify the virus in liver tissue (never previously reported), to ascertain whether this blood-transmitted virus is in fact a "hepatitis virus." SEN-V DNA prevalence was established in populations from Japan and Canada. Attempts to develop in situ hybridization, which were intended to enable us to determine whether SEN-V was infecting hepatocytes or whether it was only present in the blood perfusing the liver, were not successful. This project incorporates FY2002 projects 1Z01BP004012-07 and 1Z01BP004018-04.